Quantification of the Raf-C1 Interaction With Solid-Supported Bilayers

Abstract

By use of the quartz crystal microbalance technique, the interaction of the Raf-Ras binding domain (RafRBD) and the cysteine-rich domain Raf-C1 with lipids was quantified by using solid-supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid-supported lipid bilayers were composed of an initial octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer varying in its lipid composition as the outermost layer. The integrity of bilayer preparation was monitored by impedance spectroscopy. For binding experiments, a protein construct comprising the RafRBD and Raf-C1 linked to the maltose binding protein and a His tag, termed MBP-Raf-C1, was used. Dissociation constants and rate constants of the association and dissociation were obtained for various 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS) lipid mixtures. Independently of the phosphatidylserine (PS) content, the dissociation constants were in the order of 5x10(-7) M, while the on-rate constants were in the range of 2x10(3) (M s)(-1) and the off-rate constants in the range of 1x10(-3) s(-1). The maximum frequency shift increased significantly with increasing amounts of DMPS; this indicates that this negatively charged lipid is the primary binding site for MBP-Raf-C1. Exchange of DMPS for 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) did not alter the thermodynamics and kinetics of protein binding, which implies that the protein interaction is mainly electrostatically driven. Scanning force microscopy (SFM) was employed to render protein adsorption visible and to confirm the assumption of a protein monolayer on the lipid layer. SFM images clearly revealed that the protein binds preferentially, but not solely, to negatively charged phosphatidylserine headgroups. We hypothesize that PS-enriched domains are initial binding sites with high affinity for Raf-C1, but that lateral interactions may account for protein domain growth.

Kinetics and Thermodynamics of Annexin A1 Binding to Solid-Supported Membranes: A QCM Study

“…Interaction of Annexin A1 with Solid-Supported Membranes. Solid-supported membranes composed of an octanethiol monolayer and a second phospholipid monolayer immobilized on 5 MHz quartz plates have been proven to be well-suited for the investigation of lipid-protein interactions (29,(33)(34)(35). Here, we applied this system to investigate the interaction of full-length annexin A1 with lipid layers.…”

“…Besides these wellestablished techniques, the quartz crystal microbalance (QCM) 1 has also been recognized in recent years as a versatile and label-free technique to follow adsorption processes at solid/liquid interfaces in chemical and biological research (27,28). In combination with solid-supported lipid membranes, the quartz crystal microbalance technique allows the elucidation of thermodynamic and kinetic parameters of protein-ligand pairs in a label-free fashion due to the correspondence of frequency change to adsorbed mass (29).…”

High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes

“…As such a rapidly moving structure cannot be imaged even by high-speed AFM, it is necessary to find appropriate lipid polar groups to slow the motion. Although the relationships between the properties of protein adsorption onto planar bilayer surfaces and the lipid composition have been studied [195][196][197], we need more extensive studies on this issue.…”

Immobilization of liposomes onto quartz crystal microbalance to detect interaction between liposomes and proteins

“…Vesicle fusion and the formation of a single bilayer on a glass surface have been reported as well-established properties of neutral lipids of POPC [15][16][17]. SPBs have commonly been studied as immobilized model cell membranes, which allow the immobilization of membrane-bound enzymes, antibodies, antigens, and so on for use as biosensors [18][19][20][21][22]. On the other hand, the adsorption of intact liposomes has attracted interest only with regard to the pre-phenomena followed by vesicle fusion [12,18,23].…”